Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling

ABSTRACT

The present invention is based on a novel, alternatively spliced human isoform of MD-2 (MD-2s). In addition, the present invention relates to modified MD-2 proteins, wherein one or more tyrosine residues have been mutated to phenylalanine. In various embodiments, the invention relates to methods and kits for preventing, reducing the likelihood of developing and/or treating various conditions using MD-2s. The invention also describes methods of determining the risk of a subject to various conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of International ApplicationPCT/US09/50317, filed Jul. 10, 2009, which designated the U.S. and waspublished under PCT Article 21(2) in English, which claims the priorityof U.S. Provisional Patent Application No. 61/098,861, filed Sep. 22,2008.

This invention was made with U.S. Government support under NIAID GrantNo. AI058128 and NHLBI Grant No. HL66436. Thus, the U.S. Government mayhave certain rights in the subject matter hereof.

FIELD OF INVENTION

This invention relates to myeloid differentiation-2 and its role intoll-like receptor 4 and lipopolysaccharide signaling.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Microbial detection and instigation of an appropriate innate andsubsequent adaptive immune response to a pathogenic assault, is highlyreliant on toll-like receptors (TLRs) (Brikos et al. (2008) HANDB EXPPHARMACOL, 21-50). Members of the TLR family recognize specificconserved pathogen-associated molecular patterns (PAMPs) expressed byinvading microorganisms. TLR4, one of the most widely studied TLRs,recognizes a repertoire of PAMPs, which includes lipopolysaccharide(LPS), a major component of the outer membrane of Gram-negative bacteria(Poltorak et al. (1998) SCIENCE 282, 2085-2088; Qureshi et al. (1999) JEXP MED 189, 615-625; Hoshino et al. (1999) J IMMUNOL 162, 3749-3752;Medzhitov et al. (1997) NATURE 388, 394-397). For optimal LPS-inducedsignal transduction to occur, a receptor complex is assembled consistingof the signaling subunit TLR4, the co-receptor myeloid differentiation(MD)-2, and two accessory proteins, LPS-binding protein (LBP) and CD14(Schumann et al. (1990) SCIENCE249, 1429-1431; Pugin et al. (1993) PROCNATL ACAD SCI USA 90, 2744-2748; Frey et al. (1992) J EXP MED 176,1665-1671; Shimazu et al. (1999) J EXP MED 189, 1777-1782).

MD-2 belongs to the MD-2-related lipid recognition (ML) family (Inoharaet al. (2002) TRENDS BIOCHEM SCI 27, 219-221). A secretion signal, thesignature sequence of this group of proteins, is located at theN-terminal domain of MD-2 (Kato et al. (2000) BLOOD 96, 362-364).Although MD-2 lacks transmembrane and intracellular regions, it may bemembrane-bound through its association with the extracellular portion ofTLR4 (Akashi et al. (2000) J IMMUNOL 164, 3471-3475). Studies with micedeficient in either MD-2 or that lacked a functional TLR4 have revealedthat both proteins are absolutely required for LPS signaling (Poltoraket al. (1998) SCIENCE 282, 2085-2088; Qureshi et al. (1999) J EXP MED189, 615-625; Hoshino et al. (1999) J IMMUNOL 162, 3749-3752; Shimazu etal. (1999) J EXP MED 189, 1777-1782). Although TLR4 is critical to mounta response to gram-negative bacteria, tight regulation of the TLR4signal transduction pathway is imperative to prevent excessiveinflammation that could lead to collateral damage to the host (Liew etal. (2005) NAT REV IMMUNOL 5, 446-458). One method of control involvesalternative splicing of specific genes that encode essential componentsof the TLR4 signaling pathway to produce inhibitory isoforms, examplesinclude myeloid differentiation factor 88_(S) (MyD88_(S)) (Janssens etal. (2002) CURR BIOL 12, 467-471), and smTLR4 (Iwami et al. (2000) JIMMUNOL 165, 6682-6686; Jaresova et al. (2007) MICROBES INFECT 9,1359-1367). Similarly, the murine MD-2 gene encodes two alternativelyspliced isoforms. The truncated variant, MD-2B, generated by thesplicing out of the first 54 amino acids of exon 3, downregulates LPSsignaling (Ohta et al. (2004) BIOCHEM BIOPHYS RES COMMUN 323,1103-1108). Given that mouse and human MD-2 are highly conserved, analternative splicing of this gene in humans could also play an importantregulatory role in humans.

There is still a need for therapeutic strategies that may be used totreat human pathologies characterized by an overly exuberant or chronicimmune response to LPS. Therefore, novel mechanisms to further regulateTLR4 signaling would be beneficial and the protein MD-2s describedherein may be used to treat these human pathologies.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope.

The present invention provides a purified polypeptide comprising anamino acid sequence that is at least 80% identical to SEQ ID NO:1 or SEQID NO:2. In one embodiment, the amino acid sequence is as disclosed bySEQ ID NO:1 or SEQ ID NO:2. In another embodiment, the polypeptide isglycosylated.

In another embodiment, the amino acid sequence is as disclosed by SEQ IDNO:1 or SEQ ID NO:2 but with 1-20 conservative amino acid substitutions.Alternatively, the amino acid sequence is as disclosed by SEQ ID NO:1 orSEQ ID NO:2 but with 1-20 amino acid insertions, deletions and/orsubstitutions.

The present invention also provides an isolated nucleic acid comprisinga nucleotide sequence that encodes the polypeptide of the presentinvention.

The present invention also provides a method of inhibiting toll-likereceptor 4 signaling (“TLR4”), inhibiting lipopolysaccharide (“LPS”)signaling, treating a condition mediated by TLR4 signaling, or reducinga likelihood of developing a condition mediated by TLR4 signaling in asubject in need thereof, comprising: providing a polypeptide of thepresent invention; and administering the polypeptide to the subject toinhibit TLR4 signaling, inhibit LPS signaling, treat the conditionmediated by TLR4 signaling, or reduce the likelihood of developing thecondition mediated by TLR4 signaling. In various embodiments, thecondition mediated by TLR signaling may be selected from the groupconsisting of sepsis, septic shock, inflammation, gram negativebacterial infection, gram negative bacterial lung infection, immuneresponse such as atherosclerosis and combinations thereof.

The present invention also provides a purified polypeptide comprising anamino acid sequence of an alternatively spliced human myeloiddifferentiation-2 protein (“MD-2s”).

The present invention also provides an isolated nucleic acid comprisinga nucleotide sequence at least 80% identical to SEQ ID NO:4 or SEQ IDNO:5. In one embodiment, the isolated nucleic acid encodes a polypeptidethat binds to toll-like receptor 4 (“TLR4”) and/or or lipopolysaccharide(“LPS”).

In another embodiment, the nucleotide sequence is as disclosed by SEQ IDNO:4 or SEQ ID NO:5. In another embodiment, nucleotide sequence is adegenerate variant of SEQ ID NO:4 or SEQ ID NO:5.

The present invention also provides for an expression vector comprisinga nucleic acid of the present invention, operably linked to anexpression control sequence.

The present invention also provides a cultured cell comprising anexpression vector of the present invention.

The present invention also provides a method of producing a polypeptideof the present invention, comprising: providing a cultured cell of thepresent invention; and culturing the cell under conditions permittingexpression of the polypeptide to produce the polypeptide. In oneembodiment, the method further comprises purifying the polypeptide fromthe cell or from the medium of the cell.

The present invention also provides a nucleic acid comprising anucleotide sequence at least 80% identical to SEQ ID NO:7, SEQ ID NO:8or SEQ ID NO:9. In one embodiment, the nucleotide sequence is asdisclosed by SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In anotherembodiment, the nucleic acid hybridizes under highly stringentconditions to a region comprising exon 1 and exon 3 or a fragmentthereof of MD-2s.

The present invention also provides a method of detecting the presenceor absence of a nucleic acid that encodes an alternatively splicedmyeloid differentiation 2 (“MD-2s”) in a human subject, comprising:providing a first nucleic acid comprising a nucleotide sequence that isat least 80% identical to SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; anddetecting the presence or absence of a second nucleic acid sequence thatencodes MD-2s, wherein the presence of the second nucleic acid indicatesthe presence of MD-2s and the absence of the second nucleic acidindicates the absence of MD-2s.

In one embodiment, detecting comprises: contacting the first nucleicacid with a biological sample from the subject; and determining whetherthe first nucleic acid hybridizes under highly stringent conditions withthe second nucleic acid, wherein the presence of a binding complexcomprising the first and second nucleic acid indicates the presence ofthe second nucleic acid and the absence of the binding complex indicatesthe absence of the second nucleic acid. In another embodiment, detectingcomprises performing real time polymerase chain reaction on the sample.

The present invention also provides a method of determining a riskfactor of a subject to lipopolysaccharide (“LPS”) induced inflammationor an inflammatory disease, comprising: detecting the presence orabsence of a nucleic acid that encodes an alternatively spliced myeloiddifferentiation 2 (“MD-2s”); and correlating the presence of MD-2s witha lower risk of having the LPS induced inflammation or the inflammatorydisease or correlating the absence of MD-2s with a higher risk of havingthe LPS induced inflammation or the inflammatory disease.

In one embodiment, detecting comprises: contacting a first nucleic acidcomprising a nucleotide sequence of at least 80% identical to SEQ IDNO:7, SEQ ID NO:8, or SEQ ID NO:9 with a biological sample from thesubject; and determining whether the first nucleic acid hybridizes underhighly stringent conditions with a second nucleic acid that encodesMD-2s, wherein the presence of a binding complex comprising the firstand second nucleic acid indicates the presence of the second nucleicacid. In another embodiment, detecting comprises performing real timepolymerase chain reaction on the sample.

In one embodiment, the lipopolysaccharide induced inflammation or theinflammatory disease may be selected from the group consisting ofsepsis, septic shock, gram negative bacterial infection, gram negativebacterial lung infection, immune response such as atherosclerosis andcombinations thereof.

The present invention also provides a polypeptide consisting of an aminoacid sequence at least 80% identical to SEQ ID NO:3. In one embodiment,the amino acid sequence is as disclosed by SEQ ID NO:3. The presentinvention also provides a nucleotide sequence that encodes thepolypeptide. In one embodiment, the nucleotide sequence is as disclosedby SEQ ID NO:6.

The present invention also provides a purified polypeptide comprising anamino acid sequence comprising at least 10 consecutive amino acidresidues of SEQ ID NO:1 or SEQ ID NO:2.

In one embodiment, the amino acid sequence comprises amino acid residuesN26 to N84 of SEQ ID NO:1 or amino acid residues N10 to N68 of SEQ IDNO:2. In a particular embodiment, the amino acid sequence comprisesN-glycosylated sites at positions N26 and N84 of SEQ ID NO:1 orN-glycosylated sites at positions N10 and N68 of SEQ ID NO:2.

In another embodiment, the amino acid sequence comprises one or moreresidues involved in the dimerization interface of the TLR4/MD-2/LPScomplex. In another embodiment, the amino acid sequence comprisesresidue V52 to residue F96 of SEQ ID NO:1, or residue V36 to residue F80of SEQ ID NO:2. In another particular embodiment, the amino acidsequence comprises one or more residues selected from the groupconsisting of V52, M55, L57, R60, S88, K92, G93, 194, K95, F96 andcombinations thereof of SEQ ID NO:1; or one or more residues selectedfrom the group consisting of V36, M39, L41, R44, S72, K76, G77, 178,K79, F80 and combinations thereof of SEQ ID NO:2.

In another embodiment, the amino acid sequence comprises residue C37 toC118 involved in the second disulphide bond or a residue C65 to C75involved in the third disulphide bond of SEQ ID NO:1. In anotherembodiment, amino acid sequence comprises residue C37 and/or C118involved in the second disulphide bond or a residue C65 and/or C75involved in the third disulphide bond of SEQ ID NO:1.

In another embodiment, the polypeptide is an immunogenic polypeptidecomprising an immunogenic domain comprising at least 10 consecutiveresidues of SEQ ID NO:1 or SEQ ID NO:2.

In further embodiments, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:17 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:17. In a further embodiment, the purified polypeptideis glycosylated. In another embodiment, a purified polypeptide, theamino acid sequence of which comprises a sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17 is provided.These polypeptides may be useful for modulating LPS-induced signalingand/or TLR signaling.

In another embodiments, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:25 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:25. In a further embodiment, the purified polypeptideis glycosylated. In another embodiment, a purified polypeptide, theamino acid sequence of which comprises a sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25 is provided.These polypeptides may be useful for modulating LPS-induced signalingand/or TLR signaling.

In further embodiments, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:26 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:26. In a further embodiment, the purified polypeptideis glycosylated. In another embodiment, a purified polypeptide, theamino acid sequence of which comprises a sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25 is provided.These polypeptides may be useful for modulating LPS-induced signalingand/or TLR signaling.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts detection of an alternatively spliced isoform of MD-2 inhuman, but not mouse cells in accordance with an embodiment of thepresent invention. RNA from (A) HMECs, (B) RAW 246.7 cells, or (C)murine liver tissue, and dendritic cells were isolated and reversetranscribed into cDNA. Following cDNA synthesis, RT-PCR using human ormouse MD-2-specific primers, was performed. (D) A schematicrepresentation depicting the mature human MD-2 protein shown above thealternatively spliced MD-2s isoform. MD-2s is generated by skipping exon2. An amino acid substitution, D38G, also occurs at the junction betweenexons 2 and 3. (E) Sequence alignment of wild-type MD-2 and MD-2s. (F)Expression profiles of MD-2 and MD-2s in different human tissues. MD-2and MD-2s were amplified by RT-PCR from the indicated human tissues, asdescribed herein. (G) Detection of MD-2s in BMDMs derived fromtransgenic mice that contain human MD-2 (lanes 1, 3, and 4).

FIG. 2 shows that MD-2s is N-linked glycosylated and secreted inaccordance with an embodiment of the present invention. (A) Myc-MD-2swas immunoprecipitated (IP) from cell lysates prepared from HEK293 cellstransiently transfected with a plasmid encoding Myc-MD-2s.Immunoprecipitants were either left untreated (lane 1) or treated withpeptide N-glycosidase F (PNGase F) (lane 2). Samples were subsequentlyanalyzed by SDS-PAGE and immunoblotted with an anti-Myc antibody. (B)Myc-MD-2s was immunoprecipitated (IP) from culture supernatants thatwere obtained from HEK293 cells transiently transfected with a plasmidencoding Myc-MD-2s. Samples were subsequently analyzed by SDS/PAGE andimmunoblotted (IB) with an anti-Myc antibody. Mock transfected culturesupernatants were used as a negative control.

FIG. 3 shows that MD2-s fails to mediate either LPS-dependent NF-κBactivation or IL-8 secretion in accordance with an embodiment of thepresent invention. (A) HEK-293 cells stably transfected with TLR4 and aNF-κB reporter gene were treated with supernatants containing eitherMD-2 or MD-2s as described in the examples herein. Cells were leftuntreated or stimulated with LPS for 24 h. Mean relative stimulation ofluciferase activity±S.D. for a representative experiment, each performedin triplicate, is shown. (B) HEK-293 cells stably transfected with TLR4and a NF-κB reporter gene were transiently transfected with plasmidsencoding for either MD-2, MD-2s or CD14 as shown. 24 h later, cells wereleft untreated or stimulated with LPS (250 ng/ml) for 6 h. Mean relativestimulation of luciferase activity±SD for a representative experiment,each performed in triplicate, is shown. (C) and (D) Supernatants werealso collected and measured for IL-8 secretion.

FIG. 4 shows that MD-2s colocalizes and interacts with TLR4 inaccordance with an embodiment of the present invention. (A) Cells stablyexpressing CFP-tagged TLR4 were transiently transfected with plasmidsencoding wild-type MD-2 (upper panels) or MD-2s (lower panels). 24 hlater, cells were incubated with an anti-Myc antibody and cross-likedwith Alexa 647-conjugated anti-mouse polyclonal secondary antibody. (B)HEK293 cells were transiently transfected with a plasmid encoding forFlag-tagged TLR4 (lane 2) and cotransfected with either a Myc-taggedMD-2 (lane 3) or a Myc-tagged MD-2s (lanes 1 and 4) expressing plasmid.Co-immunoprecipitation experiments were then performed using ananti-Flag antibody (lanes 2-4) or an IgG isotype control antibody (lane1). Samples were fractionated by SDS-PAGE and immunoblotting with ananti-Myc antibody was performed. Experiment shown is representative of 4separate experiments.

FIG. 5 shows that MD-2s inhibits LPS-induced NF-κB activation inaccordance with an embodiment of the present invention. (A) HEK293 cellswere transiently transfected with plasmids encoding TLR4 and a NF-κBreporter gene in combination with MD-2 and MD-2s as indicated. 24 hlater, cells were left untreated or incubated with LPS (250 ng/ml) for 5h. Mean relative stimulation of luciferase activity±S.D. for arepresentative experiment from three separate experiments, eachperformed in triplicate, is shown. (B) HEK-293 cells stably transfectedwith TLR4 and a NF-κB reporter gene were treated with supernatantscontaining either MD-2 and MD-2s as depicted and left untreated orincubated with LPS for 24 h. Mean relative stimulation of luciferaseactivity±S.D. for a representative experiment from three separateexperiments, each performed in triplicate, is shown.

FIG. 6 shows that MD-2s interacts with wild-type MD-2 and LPS inaccordance with an embodiment of the present invention. (A) HEK293 cellswere transiently transfected with plasmids encoding for Flag-tagged MD-2(lanes 1-4) in combination with either a Myc-tagged MD-2 (lanes 1 and 3)or a Myc-tagged MD-2s (lanes 2 and 4) expressing plasmid.Co-immunoprecipitation experiments were then performed using ananti-Flag antibody (lanes 3-4) or an IgG isotype control antibody (lane1-2). Samples were fractionated by SDS-PAGE and immunoblotting with ananti-Myc antibody was performed. (B) Structural analysis of MD-2.Structural features representing the TLR4 binding sites (red); theligand contacts (dark blue); the ligand binding pocket (light blue) andthe TLR4 secondary contacts (pink) are illustrated. The residues deletedin MD-2s (yellow) are also shown. (C) HEK293 cells were transientlytransfected with plasmids encoding for Myc-tagged MD-2 (lanes 3 and 4)or a Myc-tagged MD-2s (lanes 5 and 6) expressing plasmid and treatedwith LPS-biotin as indicated. Co-immunoprecipitation experiments werethen performed using an anti-Myc antibody. Samples were fractionated bySDS-PAGE and immunoblotting with an anti-biotin (upper panel) andanti-Myc antibody (lower panel) were performed.

FIG. 7 depicts schematic diagram representing the possible mechanismsemployed by which MD-2s negatively regulates LPS signaling in accordancewith an embodiment of the present invention.

FIG. 8 shows that MD-2s inhibits lung inflammation induced by LPS invivo in accordance with an embodiment of the present invention. (A)MD-2s expression can be detected following in vivo transfection. RNA wasisolated from lung tissue of wild-type mice at 48 h post in vivotransfection with control vector (lane 1), Myc-MD2 (lane 2) or a plasmidencoding MycMD-2s (lane 3) and reverse transcribed into cDNA. RT-PCRusing human MD-2-specific primers (lanes 1-3) or murine GAPDH primers(lanes 4-6), was then performed. (B-E) Wild-type mice were transfectedwith control vector (EV, open bars) or a plasmid encoding MD-2s (blackbars) 48 h before being challenged intratracheally with PBS or 10 μg ofLPS, for 24 h. MD-2s expression reduced (B) IL-6 concentration inbronchoalveolar lavage fluid (BALF), (C) KC concentration in the lunghomogenate, and (D) recruitment of polymorphonuclear leukocytes (PMN)into the lung. (E) Flow cytometry analysis demonstrated that followingLPS challenge the percentage of GR-1⁺CD11 b⁺ cells in the lung decreasedfrom 66.7% to 26.1% with MD-2s expression.

FIG. 9 depicts the detection of MD-2s in accordance with an embodimentof the present invention. (A) MD-2 probes for real time PCR; (i) MD-2total probe recognizes both wild-type and MD-2s; (ii) MD-2s proberecognizes only the short isoform of MD-2. (B) Real-time polymerasechain reaction analysis for MD-2 expression: shown are the thresholdcycles for total MD-2 (wild-type and short isoform) and short MD-2 inepithelial cells stimulated with IFN-γ and TNF-α. The short MD-2 isoformis not expressed in unstimulated cells (flat blue line). MD-2s(blue=unstimulated; dark blue=IFN-γ; purple=TNF-α stimulation for 8 h).Total MD-2 (red=unstimulated; light blue IFN-γ; blue=TNF-α).

FIG. 10 depicts HEK293 cells transiently transfected with a plasmidexpressing MD-2s-Myc (lane 2 and 4) and subsequently treated with G418in accordance with an embodiment of the present invention. Cell lysateswere prepared (lanes 1 and 2) or culture supernatants were collected(lanes 3 and 4). Immunoprecipitations (IP) were performed using Ni-NTAbeads. Samples were subsequently analyzed by SDS/PAGE and immunoblotted(IB) with an anti-Myc antibody. HEK293 cells were used as a negativecontrol.

FIG. 11 depicts HEK293 cells that stably express MD-2 or MD-2s inaccordance with an embodiment of the present invention. HEK293 cellswere transiently transfected with plasmids expressing either MD-2-Myc orMD-2s-Myc and subsequently selected with G418. Culture supernatants fromcells stably expressing MD-2s (lanes 1 and 2) or MD-2 (lanes 3 and 4)were collected and immunoprecipitations were performed using a controlantibody (lanes 1 and 3) or with an anti-myc antibody (lanes 2 and 4).Samples were subsequently analyzed by SDS/PAGE and immunoblotted (IB)with an anti-Myc antibody.

FIG. 12 shows that MD-2 and MD-2S bind to the surface of TLR4-expressingcells in accordance with an embodiment of the present invention. HEK293Tcells stably expressing fluorescent TLR4-mCitrine were transientlytransfected with myc-tagged wild-type MD-2 or MD-2s and surface-stainedwith anti-myc antibody. (a) Cells analyzed by flow cytometry were gatedto select single cells and the TLR4-positive population. Anti-mycstaining of TLR4-mCitrine positive cells expressing MD-2 (top panel,thick line) or MD-2s (middle panel, thick line) and untransfected cells(thin line) is shown. (b) Confocal imaging shows that a subpopulation ofcells expressing mCitrine-tagged TLR4 (green) co-express transfectedMD-2 (red, top panel) or MD-2S (red, bottom panel). Co-localization ofTLR4 and MD-2 isoforms on the cell surface is visualized in yellow.

FIG. 13 shows that MD-2 is tyrosine phosphorylated and that thisphosphorylation is inhibited by herbimycin A and cytochalasin D. (A)HEK293 cells were transiently transfected with Flag-TLR4, Flag-MD-2 andCD14 constructs (lanes 2-7) or mock transfected (lane 1). 24 h latercells were left untreated (lanes 1 and 2) or stimulated with LPS (lanes3-7). Flag-tagged proteins were immunoprecipitated with an anti-Flag Abin cell lysates and analyzed by SDS-PAGE and immunoblotted with ananti-phosphotyrosine Ab (top panel), or an anti-Flag Ab (middle andlower panels). (B) HEK293 cells were transiently transfected withFlag-TLR4 and Flag-MD-2 (lanes 1-5). 24 hrs later, cells were pretreatedfor 2 hrs with herbimycin A at 0.5 μg/ml (lane 3) or 2.5 μg/ml (lane 4),or for 1 hr with 2 μM cytochalasin D (lane 5) prior to stimulation withLPS for 5 mins.

FIG. 14 shows that the mutant proteins MD-2-Y22F, MD-2-Y131F, andMD-2-Y22FY131F do not activate NF-κB as strongly as wild-type MD-2. (A)Schematic diagram showing the location of the tyrosine residues of MD-2.(B) HEK293 cells stably transfected with a NF-κB reporter gene and TLR4were transiently transfected with wild-type MD-2, MD-2-Y22F, MD-2-Y34F,MD-2-Y36F, MD-2-Y42F, MD-2-Y65F, MD-2-Y75F, MD-2-Y79F, or MD-2-Y131Fconstructs, for 24 h. Cells were left untreated or stimulated with LPSfor 6 hours and luciferase activity measured in cell lysates andexpressed as fold induction relative to mock-transfected cells (EV). (C)and (D) HEK293 cells stably transfected with a NF-κB and IL-8 reportergene and TLR4 were transiently transfected with wild-type MD-2,MD-2-Y22F, MD-2-Y131F, or MD-2-Y22FY131F constructs. Cells were leftuntreated or stimulated with LPS for 6 hours and luciferase activitymeasured in cell lysates and expressed as fold induction relative tomock-transfected cells (EV). (E) HEK293 cells were transfected withplasmids expressing the indicated mutant Flag-MD-2 proteins, wild typeFlag-MD-2 or empty vector, plus Myc-TLR4 and CD14 constructs. The cellswere stimulated with LPS for 15 minutes. Cell lysates were prepared andsamples were analyzed by immunoblotting with an p38 or ananti-phospho-p38 antibody. Flag-tagged proteins were immunoprecipitatedwith an anti-Flag Ab in cell lysates and analyzed by SDS-PAGE andimmunoblotted with an anti-phosphotyrosine Ab, or an anti-Flag Ab.

FIG. 15 depicts the structure of the TLR4-MD-2 complex. All figures arebased on the published crystal structure of the TLR4-MD-2 receptorcomplex. The structure with PDB ID: 3FXI was modified with 3-D moleculeviewer (a component of vector NTI Advance 11.0-Invitrogen). The surfacewas calculated using the Conolly method. (A) The structure of MD-2(yellow) with the location of Y131 depicted in purple. (B) The structureof MD-2 as shown in (A) in complex with TLR-4 (Cyan) and with one morecopy of the TLR-4 (blue)-MD-2 (green) complex. (C) Y131 is shown inpurple, the overall structure of MD-2 (yellow) with the location of Y22depicted in purple. (D) The structure of MD-2 as shown in (C) in complexwith TLR-4 (Cyan) and with one more copy of TLR-4 (blue)-MD-2(green)complex. The position of Y22 is shown in purple.

FIG. 16 shows that Lyn interacts with MD-2. (A) HEK293 cells weretransiently transfected with MD-2, Flag-TLR4 and CD14 construct. 24 hlater, cells were left untreated (lanes 1 and 2) or stimulated with LPS(lanes 3-5). Lyn proteins were immunoprecipitated with an anti-Lynantibody in cell lysates and analyzed by SDS-PAGE and immunoblotted withan anti-Flag Ab (top panel), or an anti-Lyn Ab (lower panel). (B) HEK293cells were transiently transfected with Flag-Lyn, Myc-TLR4 and HA-MD-2and CD14 constructs. 24 h later, cells were left untreated (lanes 1 and2) or stimulated with LPS (lanes 3-4). HA-tagged proteins wereimmunoprecipitated with an anti-HA Ab in cell lysates, and analyzed bySDS-PAGE and immunoblotted with an anti-Flag Ab. (C) HEK293 cells weretransiently transfected with Flag-Lyn and HA-MD-2 construct. 24 h later,cells were left untreated (lanes 1 and 2) or stimulated with LPS (lanes3-5). HA-tagged proteins were immunoprecipitated with an anti-HA Ab incell lysates and analyzed by SDS-PAGE and immunoblotted with ananti-Flag Ab.

FIG. 17 shows that MD-2 tyrosine phosphorylation is inhibited by Lynpeptide inhibitor. (A) HEK293 cells were transiently transfected withTLR4 and Myc-MD-2 (lanes 1-6). 24 hrs later, cells were pretreated for 2hrs with Lyn peptide inhibitor at 10, 20 or 30 μM, prior to stimulationwith LPS for 10 mins (lanes 3-6). Myc-tagged proteins wereimmunoprecipitated with an anti-Myc Ab in cell lysates, and analyzed bySDS-PAGE and immunoblotted with an anti-phosphotyrosine Ab (top panel),or an anti-Myc Ab (lower panel). (B) HEK293 cells stably transfectedwith a NF-κB or IL-8 reporter gene and TLR4 were transiently transfectedwith wild-type MD-2 24 hrs later, cells were pretreated for 2 hrs withLyn peptide inhibitor (μM) at indicated doses. Cells were left untreatedor stimulated with LPS for 6 hours and luciferase activity measured incell lysates and expressed as fold induction relative to untreatedcells.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“Therapeutically effective amount” as used herein refers to that amountwhich is capable of achieving beneficial results in a patient in need oftreatment. A therapeutically effective amount can be determined on anindividual basis and will be based, at least in part, on considerationof the physiological characteristics of the mammal, the type of deliverysystem or therapeutic technique used and the time of administrationrelative to the progression of the disease.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent, slow down and/or lessen the disease, even if thetreatment is ultimately unsuccessful.

“Conditions,” as used herein, may include, but are in no way limited toany condition or disease in which modulation of TLR4 signaling would bebeneficial, including partial or complete TLR4 inhibition. Conditionsmay include, but are in no way limited to, sepsis, septic shock,inflammation, gram negative bacterial infections, gram negativebacterial lung infections, immune responses such as atherosclerosis andother diseases or conditions resulting from or characterized by TLR4activation and/or an over-exaggerated MD2/TLR4-induced immune response.

“Isolated” nucleic acid as used herein refers to nucleic acids (e.g.,DNA or RNA) that are isolated relative to other nucleic acids in thesource material. For example, “isolated DNA” that encodes MD-2s(including cDNA) refers to DNA isolated relative to DNA which encodespolypeptides other than MD-2s.

“Purified” protein or polypeptide as used herein refer to proteins orpolypeptides that are purified relative to other proteins orpolypeptides in the source material. For example, “purified MD-2s”refers to MD-2s purified relative to proteins and polypeptides otherthan MD-2s.

“Binds specifically” as used herein refers to the act of an antibodybinding to its antigen and is intended to exclude low-level,non-specific binding that may occur between random proteins. “Bindsspecifically” as used herein is not intended and does not imply that theantibody will not bind to any protein other than the proteins orpolypeptides as disclosed herein since antibodies can cross-react withany protein that includes the relevant epitope.

An inappropriately excessive immune response causes considerablemorbidity and mortality in a number of diseases. For example, sepsis isamong the most common causes of death in the United States, with over750,000 cases presenting annually, of which more than one-quarter arefatal (Angus et al. (2001) CRIT CARE MED 29, 1303-1310). Excessiveinflammation is the hallmark of a number of related infectiouspathologies as well, including acute respiratory distress syndrome andmultiple organ failure (Miller et al. (2005) NAT REV MICRO 3, 36-46).LPS derived from bacterial sources can contribute to these diseases, anddoes so by interacting with a complex consisting of TLR4, CD14, LBP, andMD-2. To circumvent an excessive host immune response to LPS, it isimperative that TLR4 signal transduction be tightly regulated, but theprecise molecular mechanisms by which this is accomplished are onlypartly understood.

The most direct way to attenuate TLR4 signaling is by targeting specificproteins at the extracellular level, akin to the method employed by thesoluble TLR4 decoy receptors. Another mechanism dispatches the LPSreceptor complex for endosomal trafficking, which results in lysosomaldegradation of TLR4 and termination of LPS signal transduction (Husebyeet al. (2006) EMBO J 25, 683-692; Latz et al. (2002) J BIOL CHEM 277,47834-47843). In addition, the relative levels of LBP, CD14, MD-2 andTLR4 all have a direct effect on the potency of the host response to LPS(Abreu et al. (2001) J IMMUNOL 167, 1609-1616; Kitchens et al. (2003) JENDOTOXIN RES 9, 113-118). However, despite the elucidation of the MD-2and TLR4 crystalline structures (Kim et al. (2007) CELL 130, 906-917;Ohto et al. (2007) SCIENCE 316, 1632-1634), it remains unclear as to theprecise mechanism by which LPS interacts with the MD-2/TLR4 complex.While numerous studies have shown that LPS directly associates with bothTLR4 and MD-2 (Poltorak et al. (2000) PROC NATL ACAD SCI USA 97,2163-2167; da Silva Correia et al. (2001) J BIOL CHEM 276, 21129-21135),it has also been proposed that a monomeric LPS:MD-2 complex, rather thanLPS alone, is the true ligand for TLR4 (Gioannini et al. (2004) PROCNATL ACAD SCI USA 101, 4186-4191; Visintin et al. (2005) J IMMUNOL 175,6465-6472; Prohinar et al. (2007) J BIOL CHEM 282, 1010-1017).

It is clear that myeloid differentiation-2 (MD-2) is an essentialcomponent of the signaling receptor complex that recognizes andinitiates an innate immune response to bacterial LPS (Shimazu et al.(1999) J EXP MED 189, 1777-1782). At the receptor level, LPS bindingprotein (Schumann et al. (1990) SCIENCE 249, 1429-1431), CD14 (Wright etal. (1990) SCIENCE 249, 1431-1433) and TLR4 (Inohara et al. (2002)TRENDS BIOCHEM SCI 27, 219-221; Kato et al. (2000) BLOOD 96, 362-364;Visintin et al. (2001) PROC NATL ACAD SCI USA 98, 12156-12161) are thealso required in this signaling event, as is evidenced by the fact thatmice deficient in either of these proteins or MD-2 (Shimazu et al.(1999) J EXP MED 189, 1777-1782) display a similar hyporesponsiveness toLPS challenge. For signaling to occur, LPS is first extracted from thebacterial membrane by LBP, which is then transferred to CD14 in itsmonomeric form. CD14 subsequently delivers LPS to MD-2, which is asecreted glycoprotein that belongs to the MD-2-related lipid recognition(ML) family (Inohara et al. (2002) TRENDS BIOCHEM SCI 27, 219-221), thesignature sequence of which is a secretion signal. MD-2 may be presentin a soluble form or bound to the ectodomain of TLR4 (Kato et al. (2000)BLOOD 96, 362-364; Visintin et al. (2001) PROC NATL ACAD SCI USA 98,12156-12161)). Upon LPS binding, a receptor multimer composed of twocopies of the TLR4-MD-2-LPS complex is formed (Park et al. (2009) NATURE458, 1191-1195), which triggers a downstream signaling cascade,culminating in the activation of transcription factors such as nuclearfactor-κB (NF-κB) and the interferon regulatory factors (IRFs), which inturn induce various immune and inflammatory genes.

Upon ligand binding, the TLR signaling pathway initiates a cascade ofserine, threonine and tyrosine phosphorylation events. Interestingly,several members of the TLR family are also tyrosine phosphorylated,including TLR2 (Arbibe et al. (2000) NAT IMMUNOL 1, 533-540), TLR3(Sarkar et al. (2004) NAT STRUCT MOL BIOL 11, 1060-1067; Sarkar et al.(2007) J BIOL CHEM 282, 3423-3427; Sarkar et al. (2003) J BIOL CHEM 278,4393-4396), and TLR4 (Medvedev et al. (2007) J BIOL CHEM 282,16042-16053; Chen et al. (2003) AM J PHYSIOL LUNG CELL MOL PHYSIOL 284,607-613). To date the identity of the kinases involved have yet to beelucidated, however, in the case of TLR4, the Src kinase Lyn has beenimplicated in this posttranslational modification (Medvedev et al.(2007) J BIOL CHEM 282, 16042-16053). In addition to TLRs, the TLRadapter proteins, MyD88 (Ojaniemi et al. (2003) EUR J IMMUNOL 33,597-605), MyD88-adapter like (Gray et al. (2006) J BIOL CHEM 281,10489-10495), TRIF (Bin et al. (2003) J BIOL CHEM 278, 24526-24532) andTRAM (McGettrick et al. (2006) PROC NATL ACAD SCI USA 103, 9196-9201)have also been shown to be phosphorylated.

Described herein, the inventors further elucidate the complexitiesinvolved in averting an excessive and dysregulated immune response toLPS by the identification of a naturally occurring alternatively splicedisoform of human MD-2, which the inventors have termed MD-2s. Theinventors report that human MD-2s is generated by the removal of exon 2from MD-2, which leads to an in-frame deletion of 30 amino acidsspanning positions 39-69, and one amino acid substitution (D38G). Undersimilar conditions and using primers homologous to the murine MD-2 gene,a corresponding murine splice variant was not detected. The mRNAexpression profile of MD-2s revealed that it is ubiquitously expressed,suggesting that this isoform may perform a widespread role in regulatingLPS signal transduction. The inventors also detected MD-2s protein,indicating that MD-2s mRNA is not subject to nonsense-mediated decay,and found that similar to wild type MD-2, multiple forms of MD-2s weredetected upon overexpression. Glycosidase treatment established that theslower migrating forms of MD-2s represented glycosylated MD-2s protein.The glycosylation sites of MD-2 are located to the extremity of thecavity region and are believed to play a role in the secretion andstability of the protein (Ohto et al. (2007) SCIENCE 316, 1632-1634).Closer analysis of MD-2s confirmed that it is also stably secreted.

The functional studies provide significant insights into a novelregulatory mechanism employed to control TLR4 signaling upon exposure toLPS. Ectopic expression of the MD-2s isoform failed to trigger NF-κBactivation following LPS treatment, suggesting that MD-2s interfereswith normal LPS-induced signaling. Interaction studies demonstrated thatMD-2s maintains the ability to bind TLR4. This may have been predicted,given that MD-2s retains most of the residues reported to be essentialin mediating a MD-2/TLR4 interaction, with the exception of 166 and R68(Kim et al. (2007) CELL 130, 906-917; Re et al. (2003) J IMMUNOL 171,5272-5276). In addition, MD-2s interacts directly with both MD-2 andLPS. Thus, MD-2s binds to LPS, TLR4, and MD-2.

Two theoretical models of LPS-induced MD-2/TLR4 dimerization have beenproposed. Model 1 proposes that MD-2 first binds LPS, which induces astructural change in MD-2 that in turn facilitates an interactionbetween a second TLR4 molecule. In model 2, the remaining chains of LPSthat are not accommodated in the ligand binding pocket are predicted tointeract with a second TLR4 (Kim et al. (2007) CELL 130, 906-917). Anindependent study also predicts that the binding of LPS promotes TLR4homodimerization of the TLR4 ectodomains (Walsh et al. (2008) J IMMUNOL181, 1245-1254). It is thought unlikely that MD-2 homodimerization isrequired for receptor dimerization (Kim et al. (2007) CELL 130,906-917), which concurs with previous reports indicating that monomericMD-2 preferentially binds TLR4 and confers LPS responsiveness moreefficiently than MD-2 multimers (Re et al. (2002) J BIOL CHEM 277,23427-23432).

The results showing that MD-2s suppresses LPS-induced TLR4 activationare most consistent with at least two possibilities. First, MD-2s couldbe forming a complex with MD-2; this would reduce the amount of activemonomeric MD-2 available to bind both LPS and TLR4, which could in turnsuppress TLR4 activation. Secondly, MD-2s could interact directly withLPS, and then compete with the MD-2:LPS complex for binding to TLR4.These possibilities are not mutually exclusive, and either or both couldbe operative, the net effect is still the same: MD-2s suppressesLPS-induced TLR4 activation by acting as a competitive inhibitor of theactive MD2:LPS:TLR4 signaling assembly.

In addition, there is another possible mechanism by which MD-2s couldinterfere with TLR4 activation. MD-2s retains the His155 and Phe126residues, both of which are required for TLR4 dimerization. However, themissing exon may alter the tertiary structure such that these essentialresidues are no longer able to promote an effective interaction betweenMD-2s and a second TLR4 molecule, and the subsequently reduced TLR4dimerization might reduce TLR4-dependent signaling. Also this couldminimize the formation of a TLR4:MD-2 heterodimer due to the number ofTLR4:MD2 complexes being proportionally reduced as the number ofTLR4:MD-2s complexes increase.

Based on these results and while not wishing to be bound to anyparticular theory, the inventors believe that MD-2s functionally behaveslike a decoy co-receptor by binding LPS and TLR4 to form anon-functional complex that does not activate NF-κB (FIG. 7, leftpanel). In addition, MD-2s heterodimerizes with MD-2, thus decreasingthe availability of the more active monomeric MD-2, which in turn wouldbe predicted to further dampen LPS-induced NF-κB activation (FIG. 7,right panel). The inventors are currently investigating whether or notMD-2s interferes with formation of TLR4:TLR4 dimers (FIG. 7, middlepanel). Nevertheless, collectively, the results define an important rolefor MD-2s in regulating the LPS/TLR4 signal transduction pathway.

According to embodiments disclosed herein, the inventors determined therole of MD-2 in TLR4 signaling and demonstrated that MD-2 undergoestyrosine phosphorylation upon LPS stimulation and that thisphosphorylation event is required for LPS-induced NF-κB activation.Phosphorylation is a highly conserved mechanism that can be employed toregulate protein function. Indeed several studies have illustrated thatthe TLR signaling pathway is dependent on a series of phosphorylationevents. As disclosed herein, the inventors discovered that similar toTLR2, TLR3, and TLR4, MD-2 also undergoes tyrosine phosphorylation inresponse to LPS. Furthermore, the disclosed results suggest thatphosphorylation of MD-2 on specific tyrosines are required for NF-κBactivation and can be a regulatory step employed to curtail an overexuberant host immune response. Furthermore, the inventors confirmedthat this MD-2 tyrosine phosphorylation was specific to LPS stimulation,as it did not occur following stimulation with IL-1β or TNFα.

As described herein, this phosphorylation event is inhibited by thetyrosine kinase inhibitor herbimycin A. Furthermore, an endocytosisinhibitor, cytochalasin D, blocks the tyrosine phosphorylation of MD-2in cells stimulated with LPS. The inventors have identified tworesidues, located at positions 22 and 131, as possible phospho-acceptingtyrosines. Mutant proteins in which these tyrosines were altered tophenylalanine have a significantly reduced ability to activateLPS-induced NF-κB and IL-8. In addition, the inventors determined thatLyn interacts with MD2 and that a Lyn-binding peptide inhibitorspecifically abolishes MD-2 tyrosine phosphorylation, thus according tocertain embodiments Lyn is the kinase required for MD-2 tyrosinephosphorylation. The inventors have shown that MD-2 as a phosphoproteinand have demonstrated the importance of this posttranslational event asa mechanism required for MD-2-TLR4-LPS signaling.

Based on these results and while not wishing to be bound to anyparticular theory, the inventors believe that Lyn is responsible for LPSstimulated MD-2 tyrosine phosphorylation. LPS-induced tyrosinephosphorylation of MD-2 is specific, it is blocked by the tyrosinekinase inhibitor, Herbimycin A, and by an inhibitor of endocytosis,Cytochalsin-D, suggesting that MD-2 phosphorylation occurs duringtrafficking of MD2 and not on cell surface. Furthermore, the inventorshave identified two possible phospho-accepting tyrosine residues atpositions 22 and 131. Mutant proteins in which these tyrosines werechanged to phenylalanine have significantly diminished ability toactivate NF-κB in response to LPS. In addition, MD2 co-precipitates withLyn kinase and pretreatment with a Lyn-binding peptide inhibitorabolished MD2 tyrosine phosphorylation, suggesting that Lyn is a likelycandidate to be the kinase required for MD-2 tyrosine phosphorylation.The currently disclosed studies demonstrate that tyrosinephosphorylation of MD-2 is important for signaling following exposure toLPS and underscores the importance of this event in mediating anefficient and prompt immune response.

Embodiments of the present invention are based on the inventors'identification of the alternatively spliced isoform of human MD-2.Similar to wild-type MD-2, it is demonstrated herein that MD-2s issecreted and glycosylated. In addition, despite its ability to interactwith TLR4 and LPS, MD-2s fails to mediate NF-κB activation following LPSexposure. Importantly, MD-2s is identified as a negative regulator ofLPS-induced NF-κB activation. The inventors' results therefore, define anovel mechanism that can curtail excessive activation of the innateimmune response following initiation of the LPS/TLR4 signal transductionpathway.

Further embodiments of the present invention are based on the inventors'discovery that MD-2 is phosphorylated on certain tyrosine residues. Inparticular, two of the substitutions, MD-2-Y22F and MD-2-Y131F, resultedin a 50% decrease in NF-κB activity upon LPS stimulation, indicatingthat these residues were critical for maximal NF-κB activation and couldpossibly be phospho-accepting residues. Importantly, by analyzing thepublished crystal structure of MD-2, the inventors determined that thehydroxyl groups of both MD2 tyrosine residues, located at positions 22and 131, are surface exposed thereby permitting phosphorylation of theaforementioned residues to occur.

Still further embodiments of the present invention relate to the kinasefor MD-2 phosphorylation, Lyn kinase. Lyn kinase is recruited to TLR4 aswell as to CD14 upon LPS stimulation and has also been implicated as thekinase involved in the tyrosine phosphorylation of TLR4. According toembodiments described herein, MD-2 has been determined to also bepresent in a complex with TLR4 and Lyn and that MD-2 canimmunoprecipitate Lyn in the absence of CD14 and TLR4. Furthermore theinventors determined that LPS-induced MD-2 tyrosine phosphorylation isstrongly abolished following pre-treatment with a Lyn-binding peptideinhibitor. Since MD-2 and Lyn appear to directly interact with oneanother, and MD-2 tyrosine phosphorylation is diminished following Lynkinase inactivation, according to certain embodiments, that similar toTLR4, Lyn kinase is involved in the tyrosine phosphorylation of MD-2

TABLE 1 Sequences relating to human MD-2s. SEQ ID NO. SequenceDescription 1 MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYC ExpressedGRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDY proteinSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAIS GSPEEMLFCLEFVILHQPNSN 2EAQKQYWVCNSSDASISYTYCGRDLKQLYFNLYIT SecretedVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTT proteinISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVIL HQPNSN 3KMQYPISINVNPCIELKGSKGLLHIFYIP Polypeptide encoded  by Exon 2 4atgttacca tttctgtttt tttccaccct gttttcttcc Nucleotideatatttactg aagctcagaa gcagtattgg gtctgcaact sequence forcatccgatgc aagtatttca tacacctact gtgggagaga expressedtttaaagcaa ttatatttca atctctatat aactgtcaac proteinaccatgaatc ttccaaagcg caaagaagtt atttgccgaggatctgatga cgattactct ttttgcagag ctctgaagggagagactgtg aatacaacaa tatcattctc cttcaagggaataaaatttt ctaagggaaa atacaaatgt gttgttgaagctatttctgg gagcccagaa gaaatgctct tttgcttggagtttgtcatc ctacaccaac ctaattcaaa ttag 5gaagctcagaa gcagtattgg gtctgcaact catccgatgc Nucleotideaagtatttca tacacctact gtgggagaga tttaaagcaa sequence forttatatttca atctctatat aactgtcaac accatgaatc secretedttccaaagcg caaagaagtt atttgccgag gatctgatga proteincgattactct ttttgcagag ctctgaaggg agagactgtgaatacaacaa tatcattctc cttcaaggga ataaaattttctaagggaaa atacaaatgt gttgttgaag ctatttctgggagcccagaa gaaatgctct tttgcttgga gtttgtcatc ctacaccaac ctaattcaaa ttag 6ataaaatgcaatacccaatttcaattaatgttaacccctgtct Nucleotideagaattgaaaagatccaaaggattattgcacattttctacatt sequence ccaa of Exon 2

One embodiment of the present invention provides for a purified MD-2sprotein.

In one embodiment, a purified polypeptide, the amino acid sequence ofwhich comprises SEQ ID NO:1 is provided. In a particular embodiment, thepurified polypeptide consists of the amino acid sequence as set forth inSEQ ID NO:1. In a further embodiment, the purified polypeptide isglycosylated.

In another embodiment, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:2 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:2. In a further embodiment, the purified polypeptideis glycosylated.

In another embodiment, a purified polypeptide comprising 10, 20 or 30consecutive residues of SEQ ID NO:1 or SEQ ID NO:2 is provided. One ofordinary skill in the art will readily be able to screen for thepurified polypeptide's ability to modulate (e g, inhibit, induce) TLRsignaling without undue burden using methods known in the art and asdescribed herein.

In another embodiment, a purified immunogenic polypeptide comprising animmunogenic domain comprising ten consecutive residues of SEQ ID NO:1 orSEQ ID NO:2. These purified immunogenic polypeptides can be useful forproducing antibodies that bind specifically to MD-2s.

In other embodiments, the present invention provides a purifiedpolypeptide comprising residues N26 to N84 of SEQ ID NO:1 or residuesN10 to N68 of SEQ ID NO:2. In additional embodiments, the presentinvention provides a purified polypeptide comprising N-glycosylatedsites at positions N26 and N84 of SEQ ID NO:1 or N-glycosylated sites atpositions N10 and N68 of SEQ ID NO:2.

In additional embodiments, the present invention provides a purifiedpolypeptide comprising residues involved in the dimerization interfaceof the TLR4/MD-2/LPS complex. In one embodiment, the purifiedpolypeptide comprises residues V52 to F96 of SEQ ID NO:1, or residuesV36 to F80 of SEQ ID NO:2. In another embodiment, purified polypeptidecomprising one or more residues selected from the group consisting ofV52, M55, L57, R60, S88, K92, G93, 194, K95, and F96 of SEQ ID NO:1; orone or more residues selected from the group consisting of V36, M39,L41, R44, S72, K76, G77, 178, K79, and F80 of SEQ ID NO:2: (Park et al.,The structural basis of lipopolysaccharide recognition by the TLR4-MD-2complex, doi:10.1038/nature07830).

In further embodiments, the present invention provides a purifiedpolypeptide comprising residues C37 to C118, or residues C65 and/or C75.In additional embodiments, the present invention provides a purifiedpolypeptide comprising C37 and/or C118, which are residues involved inthe second disulphide bond; or C65 and/or C75, which are residuesinvolved in the third disulphide bond of SEQ ID NO:1.

In another embodiment, a purified polypeptide, the amino acid sequenceof which comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:2 is provided. In afurther embodiment, the purified polypeptide is glycosylated.

In another embodiment, a purified polypeptide comprising the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2, but with 0 to 29 conservativeamino acid substitution is provided. In particular embodiments, thepurified polypeptides have 0 to 20, 1-20, 0-10, or 1-10 conservativeamino acid substitutions. In a further embodiment, the purifiedpolypeptide is glycosylated.

In another embodiment, a purified polypeptide comprising the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2, but with 0 to 29 amino acidinsertions, deletions and/or substitutions is provided. In a particularembodiment, the purified polypeptide has 0 to 20, 1-20, 0-10, or 1-10amino acid insertions, deletions and/or substitutions. In a furtherembodiment, the purified polypeptide is glycosylated.

In another embodiment, a purified polypeptide consisting of the aminoacid sequence of SEQ ID NO:3 is provided. In another embodiment, apurified polypeptide, the amino acid sequence of which comprises asequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO:3 is provided. These polypeptides may be useful for modulatingLPS-induced signaling and/or TLR signaling.

In further embodiments, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:17 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:17. In a further embodiment, the purified polypeptideis glycosylated. In another embodiment, a purified polypeptide, theamino acid sequence of which comprises a sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17 is provided.These polypeptides may be useful for modulating LPS-induced signalingand/or TLR signaling.

In another embodiments, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:25 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:25. In a further embodiment, the purified polypeptideis glycosylated. In another embodiment, a purified polypeptide, theamino acid sequence of which comprises a sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25 is provided.These polypeptides may be useful for modulating LPS-induced signalingand/or TLR signaling.

In further embodiments, a purified polypeptide, the amino acid sequenceof which comprises SEQ ID NO:26 is provided. In a particular embodiment,the purified polypeptide consists of the amino acid sequence as setforth in SEQ ID NO:26. In a further embodiment, the purified polypeptideis glycosylated. In another embodiment, a purified polypeptide, theamino acid sequence of which comprises a sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25 is provided.These polypeptides may be useful for modulating LPS-induced signalingand/or TLR signaling.

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of MD-2s or a polypeptide asdescribed above. “Pharmaceutically acceptable excipient” means anexcipient that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic, and desirable, and includes excipientsthat are acceptable for veterinary use as well as for humanpharmaceutical use. Such excipients may be solid, liquid, semisolid, or,in the case of an aerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.“Transdermal” administration may be accomplished using a topical creamor ointment or by means of a transdermal patch. “Parenteral” refers to aroute of administration that is generally associated with injection,including intraorbital, infusion, intraarterial, intracapsular,intracardiac, intradermal, intramuscular, intraperitoneal,intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine,intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, ortranstracheal. Via the parenteral route, the compositions may be in theform of solutions or suspensions for infusion or for injection, or aslyophilized powders. Via the enteral route, the pharmaceuticalcompositions can be in the form of tablets, gel capsules, sugar-coatedtablets, syrups, suspensions, solutions, powders, granules, emulsions,microspheres or nanospheres or lipid vesicles or polymer vesiclesallowing controlled release. Via the parenteral route, the compositionsmay be in the form of solutions or suspensions for infusion or forinjection. Via the topical route, the pharmaceutical compositions basedon compounds according to the invention may be formulated for treatingthe skin and mucous membranes and are in the form of ointments, creams,milks, salves, powders, impregnated pads, solutions, gels, sprays,lotions or suspensions. They can also be in the form of microspheres ornanospheres or lipid vesicles or polymer vesicles or polymer patches andhydrogels allowing controlled release. These topical-route compositionscan be either in anhydrous form or in aqueous form depending on theclinical indication. Via the ocular route, they may be in the form ofeye drops.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentof a condition in a given subject. This amount will vary depending upona variety of factors, including but not limited to the characteristicsof the therapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Typical dosages of an effective amount of MD-2s or a polypeptide asdescribed above can be as indicated to the skilled artisan by the invitro responses or responses in animal models. Such dosages typicallycan also be reduced by up to about one order of magnitude inconcentration or amount without losing the relevant biological activity.Thus, the actual dosage can depend upon the judgment of the physician,the condition of the patient, and the effectiveness of the therapeuticmethod based, for example, on the in vitro responsiveness of therelevant primary cultured cells or histocultured tissue sample or theresponses observed in the appropriate animal models.

Another embodiment of the present invention provides for a nucleic acidor an isolated nucleic acid encoding the MD-2s protein or a polypeptideas described above.

In one embodiment, a nucleic acid, the nucleotide sequence of whichcomprises SEQ ID NO:4 is provided. In another embodiment, the nucleicacid is isolated and/or consists of SEQ ID NO:4.

In another embodiment, a nucleic acid, the nucleotide sequence of whichcomprises SEQ ID NO:5 is provided. In another embodiment, the nucleicacid is isolated and/or consists of SEQ ID NO:5.

In another embodiment, an isolated DNA, the nucleotide sequence of whichconsists of SEQ ID NO:4 or SEQ ID NO:5 is provided.

In another embodiment, a nucleic acid, the nucleotide sequence of whichcomprises a degenerate variant of SEQ ID NO:4 or SEQ ID NO:5 isprovided. In another embodiment, the nucleic acid is isolated and/orconsists of SEQ ID NO:4 or SEQ ID NO:5.

In another embodiment, an isolated nucleic acid comprising a sequencethat hybridizes under highly stringent conditions to a hybridizationprobe, the nucleotide sequence of which consists of SEQ ID NO:4, SEQ IDNO:5 or a complement of SEQ ID NO:4 or SEQ ID NO:5 is provided. In aparticular embodiment, the hybridization probe comprises SEQ ID NO:7,SEQ ID NO:8, or SEQ ID NO:9.

In another embodiment, an isolated nucleic acid comprising a sequence atleast 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4 or SEQ IDNO:5, wherein the nucleic acid encodes a polypeptide that binds to TLR4or LPS is provided. In another embodiment, the isolated nucleic acidconsists of a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO:4 or SEQ ID NO:5.

In another embodiment, an isolated nucleic acid, the nucleotide sequenceof which encodes a polypeptide at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:2, wherein thepolypeptide binds to TLR4 or LPS is provided.

In another embodiment, an isolated nucleic acid comprising a sequencethat encodes a polypeptide comprising the sequence of SEQ ID NO:1 or SEQID NO:2 with up to 10, 15, 20 or 25 conservative amino acidsubstitutions, wherein the polypeptide binds TLR4 or LPS, is provided.In a particular embodiment, the isolated nucleic acid consists of thesequence that encodes a polypeptide comprising the sequence of SEQ IDNO:1 or SEQ ID NO:2 with up to 10, 15, 20 or 25 conservative amino acidsubstitutions.

In another embodiment, an isolated nucleic acid comprising a sequencethat encodes a polypeptide comprising the sequence of SEQ ID NO:1 or SEQID NO:2 with up to 10, 15, 20 or 25 amino acid insertions, deletionsand/or substitutions, wherein the polypeptide binds to TLR4 or LPS, isprovided. In another embodiment, the isolated nucleic acid consists ofthe sequence that encodes a polypeptide comprising the sequence of SEQID NO:1 or SEQ ID NO:2 with up to 10, 15, 20 or 25 amino acidinsertions, deletions and/or substitutions.

Another embodiment provides for a nucleic acid comprising exon 2 ofhuman MD-2 gene. In one embodiment, a nucleic acid, the nucleotidesequence of which comprises SEQ ID NO:6 is provided. In anotherembodiment, the nucleic acid is isolated and consists of SEQ ID NO:6.

In another embodiment, an isolated nucleic acid comprising a sequencethat encodes a polypeptide comprising the sequence of SEQ ID NO:3 isprovided.

Another embodiment of the present invention provides for a PCR primerand/or a nucleic acid probe. The primer and/or probe can be used toidentify the presence or absence of MD2s. In one embodiment, the PCRprimer and/or nucleic acid probe is a nucleic acid comprising SEQ IDNO:7, SEQ ID NO:8, or SEQ ID NO:9. In another embodiment, the PCR primerand/or nucleic acid probe comprises a nucleic acid at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical SEQ ID NO:7, SEQ ID NO:8, orSEQ ID NO:9. In another embodiment, the PCR primer and/or nucleic acidprobe hybridizes under highly stringent conditions to a regioncomprising exon 1 and exon 3 or a fragment thereof of MD-2s. In oneparticular embodiment, the PCR primer and/or nucleic acid probehybridizes under highly stringent conditions to about 20 nucleotides ofexon 1 and about 20 nucleotides of exon 3. In other particularembodiments, the PCR primer and/or nucleic acid probe hybridizes underhighly stringent conditions to about 10 to 20 nucleotides of exon 1 andabout 10 to 20 nucleotides of exon 3. In other particular embodiments,the PCR primer and/or nucleic acid probe hybridizes under highlystringent conditions to about 15 to 20 nucleotides of exon 1 and about15 to 20 nucleotides of exon 3. In another particular embodiment, thePCR primer and/or nucleic acid probe hybridizes under highly stringentconditions to about 15, 14, 13, 12, 11 or 10 nucleotides of exon 1 andabout 15, 14, 13, 12, 11 or 10 nucleotides of exon 3.

In another embodiment, an expression vector comprising a nucleic acid asdescribed above operably linked to an expression control sequence isprovided. In various embodiments, the expression vector and expressioncontrol sequence may be any expression vector or expression controlsequence known in the art or as described herein. Examples of expressionvectors include but are not limited to pCDNA3.1, pCMV-HA and pEF-BOS.Examples of expression control sequences include but are not limited tohuman cytomegalovirus immediate early (CMV) promoter and EF1-α promoterregion.

In another embodiment, a cultured cell comprising the expression vectorcomprising a nucleic acid as described above is provided.

In another embodiment, a cultured cell comprising a nucleic acid asdescribed above, operably linked to an expression control sequence isprovided. In various embodiments, the expression control sequence may beany expression control sequence known in the art or as described herein.

In another embodiment, a cultured cell transfected with an expressionvector comprising a nucleic acid as described above or a progeny of saidcell, wherein the cell expresses a polypeptide as described above isprovided.

Another embodiment of the present invention provides for a method ofproducing MD-2s or a polypeptide as described above is provided.

In one embodiment, the method comprises culturing a cultured celldescribed above under conditions permitting expression of thepolypeptide. In a further embodiment, the method comprises purifying thepolypeptide from the cell or the medium of the cell.

In another embodiment, the method comprises culturing a cultured celldescribed above under conditions permitting expression under the controlof the expression control sequence, and purifying the polypeptide fromthe cell or the medium of the cell.

Another embodiment of the present invention provides for methods ofusing the isolated MD-2s protein, or compositions comprising theisolated MD-2s protein.

In one embodiment, the method is for inhibiting TLR4 signaling in asubject in need thereof. The method comprises providing a purified MD-2sprotein or polypeptide as described above and administering the purifiedMD-2s protein or polypeptide as described above to the subject. Thepurified MD-2s protein or polypeptide as described above may be in acomposition that further comprises a pharmaceutically acceptable carrieror a pharmaceutically acceptable excipient.

In one embodiment, the method is for inhibiting LPS signaling in asubject in need thereof. The method comprises providing a purified MD-2sprotein or polypeptide as described above and administering the purifiedMD-2s protein or polypeptide as described above to the subject. Thepurified MD-2s protein or polypeptide as described above may in acomposition that further comprises a pharmaceutically acceptable carrieror a pharmaceutically acceptable excipient.

In another embodiment, the method is for treating a condition mediatedby TLR4 signaling or reducing the likelihood of developing a conditionmediated by TLR signaling in a subject in need thereof. The methodcomprises providing a purified MD-2s protein or a polypeptide asdescribed above and administering the purified MD-2s protein orpolypeptide as described above to the subject. The purified MD-2sprotein or polypeptide may be in a composition that further comprises apharmaceutically acceptable carrier and/or a pharmaceutically acceptableexcipient. In various embodiments, the condition is sepsis, septicshock, inflammation, gram negative bacterial infection, gram negativebacterial lung infection and/or immune response such as atherosclerosis.

Another embodiment of the present invention provides a method ofdetecting the presence or absence of a nucleic acid that encodes MD-2sin a subject. The method comprises providing a PCR primer or nucleicacid probe as described above; and detecting the presence or absence ofa second nucleic acid that encodes MD-2s in a biological sample of thesubject, wherein the presence of the second nucleic acid indicates thepresence of MD-2s and the absence of the second nucleic acid indicatesthe absence of MD-2s. In one embodiment, the method comprises providinga first nucleic acid, the nucleotide sequence of which comprises SEQ IDNO:7, SEQ ID NO: 8, or SEQ ID NO:9; and detecting the presence orabsence of a second nucleic acid that encodes MD-2s in a biologicalsample of the subject, wherein the presence of the second nucleic acidindicates the presence of MD-2s and the absence of the second nucleicacid indicates the absence of MD-2s. These methods may further comprisereporting the presence or absence of the second nucleic acid. Forexample, a laboratory performing the test may report the results to amedical practitioner, such as the subject's doctor and/or the subject'sdoctor may report the results to the subject. In other non-limitingexamples, reporting may comprise recording the results on an electronicstorage medium, displaying the results on a computer screen, or printingthe results on a piece of paper.

Examples of “biological sample” include but are not limited to mammalianbody fluids, sera such as blood (including whole blood as well as itsplasma and serum), CSF (spinal fluid), urine, sweat, saliva, tears,pulmonary secretions, breast aspirate, prostate fluid, seminal fluid,stool, cervical scraping, cysts, amniotic fluid, intraocular fluid,mucous, moisture in breath, animal tissue, cell lysates, tumor tissue,hair, skin, buccal scrapings, nails, bone marrow, cartilage, prions,bone powder, ear wax, etc. or even from external or archived sourcessuch as tumor samples (i.e., fresh, frozen or paraffin-embedded).

In one embodiment, detecting the presence or absence of the secondnucleic acid comprises contacting the PCR primer or nucleic acid probewith a biological sample of the subject and performing real timepolymerase chain reaction on the sample. In a particular embodiment,detecting the presence or absence of the second nucleic acid comprisescontacting the first nucleic acid with a biological sample of thesubject and performing real time polymerase chain reaction on thesample.

In another embodiment, detecting the presence or absence of the secondnucleic acid comprises contacting the PCR primer or nucleic acid probewith a biological sample from the subject and determining whether thePCR primer or nucleic acid probe hybridizes under highly stringentconditions with the second nucleic acid in the biological sample,wherein the presence of a first and second nucleic acid binding complexindicates the presence of the second nucleic acid. In a particularembodiment, detecting the presence or absence of the second nucleic acidcomprises contacting the first nucleic acid with a biological samplefrom the subject and determining whether the first nucleic acidhybridizes under highly stringent conditions with the second nucleicacid in the biological sample, wherein the presence of a first andsecond nucleic acid binding complex indicates the presence of the secondnucleic acid.

Individuals who do not have MD-2s may have a higher risk of developingLPS-induced inflammation or an inflammatory disease. For example, anindividual without MD-2s may develop severe sepsis when they areinfected with gram negative bacteria. Therefore, another embodiment ofthe present invention provides for a method to determine a subject'srisk of developing LPS-induced inflammation or an inflammatory disease.In one embodiment, the method comprises detecting the presence orabsence of a nucleic acid that encodes MD-2s or a polypeptide asdescribed above; and correlating the presence of the nucleic acid with alower risk of developing LPS-induced inflammation or an inflammatorydisease or correlating the absence of the second nucleic acid with ahigher risk of developing LPS-induced inflammation or an inflammatorydisease. These methods may further comprise reporting the presence orabsence of the nucleic acid that encodes MD-2s, or reporting the low orhigh level of risk of developing LPS-induced inflammation aninflammatory disease. For example, a laboratory performing the test mayreport the results to a medical practitioner, such as the subject'sdoctor and/or the subject's doctor may report the results to thesubject. In other non-limiting examples, reporting may compriserecording the results on an electronic storage medium, displaying theresults on a computer screen, or printing the results on a piece ofpaper. Examples of LPS-induced inflammation or an inflammatory diseaseinclude but are not limited to sepsis, septic shock, gram negativebacterial infection, gram negative bacterial lung infection and immuneresponse such as atherosclerosis.

Alternatively, the method comprises detecting the expression level of anucleic acid that encodes MD-2s or a polypeptide as described above;comparing the expression level of the nucleic acid to a standardizedexpression level of a nucleic acid that encodes MD-2s determined fromindividuals who have MD-2s; and correlating an expression level that isequal or higher than the standardized expression level to a lower riskof developing LPS-induced inflammation or an inflammatory disease orcorrelating an expression level that is lower than the standardizedexpression level to a higher risk of developing LPS-induced inflammationor an inflammatory disease. These methods may further comprise reportingthe low or high level of risk of developing LPS-induced inflammation aninflammatory disease. For example, a laboratory performing the test mayreport the results to a medical practitioner, such as the subject'sdoctor and/or the subject's doctor may report the results to thesubject. In other non-limiting examples, reporting may compriserecording the results on an electronic storage medium, displaying theresults on a computer screen, or printing the results on a piece ofpaper. Examples of LPS-induced inflammation or an inflammatory diseaseinclude but are not limited to sepsis, septic shock, gram negativebacterial infection, gram negative bacterial lung infection and immuneresponse such as atherosclerosis.

The present invention describes kits for preventing or reducing thelikelihood of developing and/or treating a condition using MD-2s andkits for determining a subject's risk of developing severe sepsis.Various embodiments of the present invention thus provides for kits forpreventing or reducing the likelihood of developing and/or treating acondition in a mammal in need thereof, comprising: a quantity of MD-2sor a polypeptide as described above and instructions for administeringthe quantity of MD-2s or the polypeptide as described above to themammal to prevent, reduce the likelihood of developing and/or treat thecondition. Another embodiment of the present invention provides for akit for determining a subject's risk of developing these diseases ordisease conditions, comprising: a quantity of a PCR primer or nucleicacid probe as described above; and instructions for using the quantityPCR primer or nucleic acid probe to determine the subject's risk ofdeveloping these diseases or disease conditions. References herein toMD-2s include synthetic, recombinant, naturally-occurring and any otherforms of the protein. In one embodiment, the condition is sepsis orseptic shock. In another embodiment, the condition is inflammation. Inanother embodiment, the condition is a lung infection.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Biological Reagents and Cell Culture

Immortalized human dermal microvessel endothelial cells (HMEC) (a kindgift from F. J. Candal, Center for Disease Control and Prevention,Atlanta, Ga.) were cultured in MCDB-131 medium, supplemented with 10%heat-inactivated fetal bovine serum, 2 mM glutamine, and 100 μg/mlpenicillin and streptomycin. The HEK293 cell line and the mouse RAW264.7 macrophage cell line were cultured in Dulbecco's modified Eagle'smedium, supplemented with 10% heat-inactivated FBS and 2 mM glutamine.The plasmid encoding human wild-type Flag MD-2 was a generous gift fromKensuke Miyake. LPS (TLRGrade) was from (Alexis) and biotin-LPS(Ultrapure) was from Invivogen. Protein tyrosine kinase inhibitorherbimycin A was purchased from Sigma-Aldrich. Lyn peptide inhibitor waspurchased from Tocris Cookson. 4G10 anti-phosphotyrosine Ab waspurchased from Upstate. Anti-Flag agarose affinity gel and anti-Flag Abwere from Sigma Aldrich. Anti-Myc and Anti-HA Abs were from Santa CruzBiotechnology and Zymed Labs respectively.

Example 2 RT-PCR

Total cellular RNA was isolated from HMEC, RAW 264.7, murine dendriticcells, and murine liver tissue using RNeasy mini kit (Qiagen, Valencia,Calif.). RNA from human lung, pancreas, thymus, kidney, spleen, liver,heart, and placenta was purchased from Ambion (Austin, Tex.). Followingreverse transcription with Omniscript cDNA synthesis kit (Qiagen), PCRanalysis was performed using primers specific for the human MD-2 (sense:ATGTTACCATTTCTGTTT (SEQ ID NO:10)), antisense: CTAATTTGAATTAGGTTG (SEQID NO:11)) or mouse MD-2 (sense: TCTGCAACTCCTCCGATG (SEQ ID NO:12),antisense: GGCGGTGAATGATGGTGA (SEQ ID NO:13)). The PCR was performedusing Taq DNA polymerase (Invitrogen, Carlsbad, Calif.).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loadingcontrol.

Example 3 Immunoprecipitation and Immunoblotting

HEK 293 cells were seeded into 100 mm dishes (1.5×10⁶) 24 h prior totransfection. Transfections were performed according to themanufacturer's instructions using lipofectamine. Forco-immunoprecipitations, 4 μg of each construct was transfected. Forcompetition experiments where the effect of increasing sMD-2 expressionon complex formation between two signaling molecules was examined, 2 μgof each signaling molecule expression plasmid was transfected in thepresence of increasing amounts of the sMD-2 expression plasmid. Thetotal amount of DNA in each sample was kept constant by using emptyvector cDNA. In all cases, cells were harvested 24 h followingtransfection in 600 μl of lysis buffer (50 mM HEPES, pH 7.5, 100 mMNaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 containing protease inhibitorcocktail, and 1 mM sodium orthovanadate). For immunoprecipitations, theindicated antibodies were incubated with the cell lysates overnight at4° C. Subsequently, Trueblot™ IgG beads were added and the samples wereincubated at 4° C. for 1 hr. The immune complexes were then washed andthe associated proteins were eluted from the beads by boiling in 35 μlof sample buffer, and then fractionated by SDS-PAGE. For immunoblotting,primary antibodies were detected using horseradish peroxidase-conjugatedsecondary antibodies, followed by enhanced chemiluminescence (AmershamBiosciences).

Example 4 NF-κB and IL-8 Promoter Luciferase Activation in HEK 293 Cells

HEK293 cells were transiently transfected with the expression vectorsnoted in combination with constructs encoding the NF-κB- andIL-8-luciferase reporter gene, and either the β-galactosidase gene orthe phRL-TK report gene to normalize for transfection efficiency. In allcases, total DNA concentration was kept constant by supplementation withempty vector control. Following overnight incubation, cells werestimulated for 6 hours with 50 ng/ml LPS and then lysed, and luciferaseactivity was measured as described previously (Ohta et al. (2004)BIOCHEM BIOPHYS RES COMMUN 323, 1103-1108). Data are shown as mean±SD ofthree or more independent experiments and are reported as a percentageof LPS-stimulated NF-κB and IL-8 promoter activity, or relativeluciferase activity.

Example 5 Identification of a Novel Human MD-2 Splice Variant

During the inventors' analysis of MD-2 expression in HMECs, two cDNAproducts approximately 390 and 480 bp in length (FIG. 1A) were detectedby RT-PCR. The expression of murine MD-2 was also examined by performingRT-PCR on cDNA derived from the murine cell line, RAW 246.7 (FIG. 1B).In contrast, using primers homologous to the same region in the mouse asin the human, only the larger RT-PCR product was detected. To confirmthat the absence of the smaller fragment was not specific to the murinecell line selected, cDNA from murine bone marrow derived dendritic cellsand liver tissues obtained from C57BL/6 mice were also amplified. Againonly the larger cDNA fragment was observed in the murine tissues tested(FIG. 1C).

Upon sequencing the larger cDNA fragment detected in HMECs, it wasdetermined that this PCR product corresponded to the published sequenceof full-length human MD-2. Sequence analysis of the smaller cDNAfragment (subsequently referred to as MD-2s), revealed that this was asplice variant of human MD-2, which lacked the region encoded by exon 2of the MD-2 gene (FIGS. 1D and E). This isoform is putatively translatedinto a 114-residue protein with no frame shift, however, one amino acidsubstitution, D38G, occurs at the junction between exons 2 and 3.

To further characterize the expression profile of MD-2s, RT-PCR analysison a variety of human tissues was performed. As shown in FIG. 1G, MD-2swas widely expressed in all human tissues analyzed. Furthermore, it wasobserved that the full length wild-type MD-2 is the predominant formdetected, although the ratio between MD-2 and MD-2s varies in differenttissues. These findings establish that MD-2s is not a result oftissue-specific alternative splicing, but rather is ubiquitouslyexpressed in all human tissues tested.

Having established that MD-2s is not expressed in the mouse, it wasinvestigated if MD-2s could be detected in transgenic miceoverexpressing the human MD-2 gene. RT-PCR analysis conducted on BMDMsfrom these mice revealed that MD-2s can be detected irrespective of thepresence or absence of human or murine TLR4 (FIG. 1G, compare lane 3, tolane 1 or 4).

To determine why the human but not the mouse MD-2 gene alternativelyskips exon 2, the gene structures and sequences of the two species werecompared (Table 2). Both the human and the murine MD-2 genes areorganized into five exons and four introns, and each species encodes apredicted full-length MD-2 protein of 145 amino acids. Alignment of thehuman and mouse genomic regions revealed that the exons and codingsequences were well conserved. However, analysis of the non-codingregions revealed a number of differences. In particular, it was notedthat intron 1 of human MD-2 is composed of 13239 base pairs, while themouse has 3067. The longer intron 1 may increase the probability foralternative lariat formation during the splicing process of human MD-2.In addition, after comparing the sequences at the 3′ end of intron 1,murine MD-2 was found to have more pyrimidines than human MD-2, whichmay lead to a more stable lariat formation in murine MD-2, therebypreventing alternative splicing of exon 2 in this species.

TABLE 2 E-1   E5- coding Intron 1 Sequences at 3' of Intron 1 E2 E3 E4coding (a) Human 112 13241 ttgacattatctttattgcttttag 90 129 53 99(SEQ ID NO: 14) Mouse 112 3064 ttg-TattTtcttCattCcttttag 90 129 53 99(SEQ ID NO: 15) (b) 5′-ttgacattatctttattgatttagATAAAATGCAATACCCAATTTCAATTAATGTTAACCCCTGTCTAGAATTGAAAAGATCCAAAGGATTATTGCACATTTTCTACATTCCAA gt aagttcaaatttttgcttttata-3′ (SEQ ID NO: 16) (a) Comparison of human and mouseMD-2 gene structure. (b) The intron-exon boundary of exon 2 in the MD-2gene is depicted, with the intronic sequences in lower case and theexcised sequence in upper case.

Example 6 Similar to Wild-Type MD-2, MD-2s is Glycosylated and Secreted

In order to characterize this newly identified isoform of human MD-2,the smaller RT-PCR fragment was amplified and cloned into an expressionvector containing a Myc tag. HEK293 cells were subsequently transientlytransfected with MD-2s-Myc. Cellular extracts were later prepared andSDS-PAGE analysis was performed. Similar to wild-type MD-2, multipleforms of MD-2s could be detected upon overexpression (FIG. 2A, lane 1).The altered electrophorectic mobility of wild-type MD-2 is due toN-linked glycosylations at positions Asn26 and Asn114 (Ohnishi et al.(2001) J IMMUNOL 167, 3354-3359). These residues are still present inMD-2s, but since MD-2s lacks 30 amino acids, the tertiary structure islikely to be different from wild-type MD-2, which could result inocclusion of these known glycosylation sites. To determine if thealtered electrophorectic mobility of MD-2s was due to glycosylation,immunoprecipitated MD-2s, isolated from HEK293 cells transientlyexpressing MD-2s-Myc, was treated with the N-glycosidase, PNGaseF.Samples were then analyzed by SDS-PAGE and immunoblotted with ananti-Myc antibody. It was found that the slowest migrating forms ofMD-2s were no longer evident in the PNGaseF-treated sample (FIG. 2A,lane 2). These results indicate that similar to wild type MD-2, MD-2s isa glycoprotein.

Next, it was investigated if MD-2s exists as a stably secreted protein.Myc-tagged proteins were immunoprecipitated from culture supernatantsobtained from HEK293 cells transiently expressing MD-2s-Myc. Sampleswere subsequently analyzed by SDS-PAGE and immunoblotted with ananti-Myc antibody. As shown in FIG. 2B, lane 2, MD-2s is present in asoluble form.

Example 7 MD-2s Fails to Induce NF-κB Activation Following LPSStimulation

The requirement for MD-2s in LPS mediated NF-κB activation was assessed.It has been shown that the soluble form of wild-type MD-2 confers LPSresponsiveness to cells expressing TLR4; therefore, it was investigatedif soluble MD-2s was also bioactive. Culture supernatants were obtainedfrom HEK293 cells transiently expressing control vector, MD-2, or MD-2s,and incubated with HEK293 cells stably transfected with TLR4 and anNF-κB-dependent luciferase reporter gene. As previously shown, solubleMD-2 strongly activated NF-κB following LPS stimulation (FIG. 3A).However, the secreted form of MD-2s could not confer LPS responsivenessto these cells as measured by NF-κB activation (FIG. 3A) or IL-8secretion (FIG. 3C).

MD-2s was tested to see if it needed to be transiently expressed in TLR4reporter cells in order to mediate NF-κB activation following LPSstimulation. HEK293 cells stably transfected with TLR4 and anNF-κB-dependent luciferase reporter gene were transiently transfectedwith plasmids encoding CD14 in combination with either MD-2 or MD-2s,and subsequently treated with LPS. In agreement with published results,wild-type MD-2 activated NF-κB in response to LPS treatment. Incontrast, MD-2s was unable to induce LPS-mediated NF-κB activation, evenat higher concentrations (FIG. 1B) or IL-8 secretion (FIG. 1D). Takentogether, these results suggest that the region of human MD-2 encoded byexon 2 plays an important role in mediating LPS-induced signaling.

Example 8 Similar to Wild-Type MD-2, MD-2s Interacts with TLR4

Given that MD-2s failed to induce LPS-dependent NF-κB activation, theinventors questioned whether this might be due to an inherent inabilityto interact directly with TLR4. To address this possibility,co-localization studies to examine the cellular distribution of MD-2swith respect to TLR4 were performed. HEK293 cells stably expressingTLR4CFP were subsequently transiently transfected with a plasmidencoding either MD-2 (FIG. 4A, upper panel) or MD-2s (FIG. 4A, lowerpanel) and stained with an anti-Myc antibody. Confocal analysis revealedthat both TLR4 and MD-2s exhibited the same cellular distribution (FIG.4A, lower right hand side panel). Next whether MD-2s is physicallyassociated with TLR4 was assessed. HEK293 cells were transientlytransfected with a plasmid encoding FLAG-tagged TLR4 in combination witheither a Myc-tagged MD-2 or a Myc-tagged MD-2s expressing plasmid, andco-immunoprecipitation experiments were performed. It was determinedthat similar to MD-2, a direct interaction between MD-2s and TLR4 wasobserved (FIG. 4B, lane 4), indicating that although MD-2s fails toinduce LPS-dependent NF-κB activation, this cannot be attributed to aninability to associate with TLR4.

Example 9 MD-2s Diminishes LPS-Induced NF-κB Activation

Having determined that MD-2s does not activate NF-κB, we nextinvestigated the effect of MD-2s on MD-2/TLR4-mediated LPS signaling.HEK293 cells were transiently transfected with plasmids encoding CD14,TLR4, and wild-type MD-2 in conjunction with a plasmid encoding theNF-κB-dependent luciferase reporter gene in the presence or absence of aplasmid encoding MD-2s. As shown in FIG. 5A, MD-2s significantlyinhibited LPS-induced NF-κB activation. Furthermore, we assessed theinhibitory role of soluble MD-2s following LPS stimulation. HEK293 cellsstably transfected with TLR4 and an NF-κB-dependent luciferase reportergene were incubated with supernatants obtained from HEK293 cellstransiently expressing a constant amount of MD-2 with increasingconcentrations of MD-2s. It was determined that soluble MD-2s inhibitsNF-κB activation (FIG. 5B). This suggests that the region encoded byexon 2 is critical for efficient TLR4 signaling.

Example 10 MD-2s Interacts with MD-2 and LPS

Previous reports indicate that monomeric MD-2 preferentially binds TLR4and confers LPS responsiveness more efficiently than MD-2 multimers (Reet al. (2002) J BIOL CHEM 277, 23427-23432). Therefore, it wasinvestigated if MD-2s could interact with wild-type MD-2, which couldpotentially diminish responsiveness to LPS by reducing the amount ofavailable monomeric MD-2. HEK293 cells were transiently transfected witha plasmid encoding FLAG-tagged MD-2, in combination with either aMyc-tagged MD-2 or a Myc-tagged MD-2s expressing plasmid, andco-immunoprecipitation experiments were performed. Similar to previousreports, it was observed that MD-2 can homodimerise (FIG. 6A, lane 2).It was also noted that MD-2s associates with wild-type MD-2 (FIG. 11,lane 3). These results raise the possibility that the mechanism by whichMD-2s inhibits LPS-induced TLR4-dependent signaling is by reducing theamount of the active monomeric species of MD-2, and thus increasing themultimeric form of MD-2. Both would be predicted to lead to a reductionin TLR4-dependent signaling during exposure to LPS (Re et al. (2002) JBIOL CHEM 277, 23427-23432; Teghanemt et al. (2008) J BIOL CHEM 283,21881-21889).

The published structural models of MD-2 were also analyzed to ascertainthe structural effect of deleting exon 2. MD-2 consists of a β-cup foldwith two anti-parallel β-sheets, the first one composed of six β-strands(numbered 1/2/9/8/5/6) and the other of three (numbered 3/4/7) (Kim etal. (2007) CELL 130, 906-917; Ohto et al. (2007) SCIENCE 316,1632-1634). The missing exon 2 of MD-2s encodes the first two β-strandsof the three-stranded β-sheet (β3 and β4 strands) (FIG. 6B). The hingesconnecting β-strands 2 to 3 and 4 to 5 are also partially lost.Furthermore, the disulphide bond between Cys25 and Cys51, which assistsin closing the MD-2 cavity and stabilizing the cup-like structure, isdisrupted. The β6 and β7 strands that line the entrance to the deephydrophobic cavity are still encoded by MD-2s mRNA.

Previous mutational studies have demonstrated that the LPS-bindingregion of MD-2 depends on Lys89, Arg90, Lys91, Phe119, Phe121, Lys122,Lys125, Lys128, and Lys132. Several of these residues have also beenshown to be directly involved in the binding of Eritoran and Lipid IVato MD-2 (Kim et al. (2007) CELL 130, 906-917; Ohto et al. (2007) SCIENCE316, 1632-1634). Although all of the aforementioned residues are presentin MD-2s, structural analysis of this protein implied that theligand-binding pocket may be severely disrupted, suggesting that MD-2smay be unable to bind LPS efficiently. To address this possibility,HEK293 cells were transiently transfected with plasmids encoding eitherMD-2 or MD-2s. Culture supernatants were then incubated with biotin-LPSand Myc-tagged proteins were immunoprecipitated. In agreement withprevious studies, secreted MD-2 bound readily to LPS (FIG. 6C, lane 4)(Visintin et al. (2003) J BIOL CHEM 278, 48313-48320). Interestingly,under similar conditions, an interaction between secreted MD-2s and LPSwas also detected (FIG. 6C, lane 6), suggesting that exogenous MD-2s mayactively sequester LPS from MD-2. Hence, these results suggest a secondway that MD-2s could suppress LPS-induced activation of TLR4 is bydirectly binding LPS, thereby reducing the availability of LPS to bindwith monomeric MD-2 and thus diminishing TLR4 signal transduction.

Example 11

Real-time polymerase chain reaction analysis for MD-2s expression wasperformed on epithelial cells. The primers/probes for MD-2s are as shownin Table 3. Results are shown in FIG. 9 b.

TABLE 3  Sense 5′-ATTGGGTCTGCAACTCATCC-3′ (SEQ ID NO: 7) Antisense5′-CGCTTTGGAAGATTCATGGT-3′ (SEQ ID NO: 8) Probe5′-CCTACTGTGGGAGAGATTTAAAG-3′ (SEQ ID NO: 9)

Example 12 Flow-Cytometric Analysis of MD-2 and MD-2s Expression

HEK293T cells were retrovirally transduced to generate a cell linestably expressing TLR4-mCitrine. TLR4-mCitrine cells in 12-well disheswere transfected with 300 ng of plasmid encoding myc-tagged MD2 or MD2Susing GeneJuice lipofection reagent (Novagen) and cultured overnight.Cells were dislodged by scraping, washed once with cold PBS andincubated with a 1:50 dilution of anti-myc Alexa 647-conjugated antibody(AbD Serotec) at 4° C. for 30 minutes. Cells were washed four times withPBS for and fluorescence was assessed with an LSR II flow cytometer (BDBiosciences) running FACS Diva software (BD Biosciences). Data wasprocessed using FlowJo software v8.6.3 (Tree Star).

Example 13 Confocal Imaging of MD-2 and MD-2s Transfected Cells

TLR4-mCitrine cells were cultured on glass-bottom confocal dishes(MatTek) coated with collagen. After 24-hours of culture, cells weretransfected with up to 300 ng of plasmid encoding myc-tagged MD-2 orMD-2S using GeneJuice lipofection reagent (Novagen) and cultured forapproximately 24 hours. Cells were stained with anti-myc Alexa-647antibody (AbD Serotec) in culture medium at 4° C. for 20 minutes. Cellswere gently washed 3 times with PBS and culture medium was replaced.Cells were imaged at room temperature using an SP2 AOBS confocal laserscanning microscope (Leica Microsystems) running LCS software (LeicaMicrosystems).

Example 14 MD-2 was Found to be Tyrosine Phosphorylated Upon Stimulationwith LPS

Tyrosine phosphorylation of numerous proteins is induced uponstimulation with LPS and indeed the LPS receptor, TLR4, is itselftyrosine phosphorylated. Given the important role of MD-2 following LPSstimulation, the inventors investigated whether MD-2 ispost-translationally modified as well. HEK293 cells were transientlytransfected with plasmids expressing Flag-TLR4, Flag-MD-2 and CD14 ormock transfected. 24 hrs later, cells were left untreated or stimulatedwith LPS for various time points. Proteins were immunoprecipitated fromcell extracts with an anti-Flag Ab and analyzed by immunoblotting withan Ab that detects proteins phosphorylated on phosphotyrosine residues.Upon stimulation with LPS, MD-2 was found to be tyrosine phosphorylated(FIG. 13A). The presence of 5 mM phosphotyrosine, a competitiveinhibitor, completely abrogated the immunoreactivity detected by theanti-phosphotyrosine Ab, in contrast, phosphoserine or phosphothreoninehad no effect on MD-2 tyrosine phosphorylation (data not shown), whichconfirms the specificity of this result. In addition, MD-2 tyrosinephosphorylation was not observed after stimulation with IL-β3 or TNFα orRsDPLA, a biologically inactive analogue of lipid A (data not shown). Tofurther confirm that MD-2 is posttranslationally modified, the inventorspre-treated HEK 293 cells, overexpressing TLR4 and MD-2, with thetyrosine kinase inhibitor herbimycin A for two hours prior to LPSstimulation and observed that herbimycin A significantly inhibitedLPS-induced MD-2 tyrosine phosphorylation in a dose dependent manner(FIG. 13B, compare lanes 3 and 4 to lane 2).

Next, the inventors investigated whether MD-2 phosphorylation occurredduring trafficking, instead of on the cell surface. More specifically,the inventors examined the role of receptor or ligand internalizationand endocytosis on the phosphorylation status of MD-2. Sincecytochalasins effectively block LPS internalization and signaling forcytokine release (Poussin et al. (2006) J BIOL CHEM 273, 20285-20291),HEK293 cells, overexpressing TLR4 and MD-2, were pre-treated withCytochalasin-D for one hour prior to LPS stimulation. The inventorsobserved that LPS-induced MD-2 tyrosine phosphorylation wassignificantly inhibited following pretreatment with cytochalasin D (FIG.13B, lane 5). Thus according to certain embodiments MD-2 tyrosinephosphorylation occurs intracellularly during trafficking and not on thecell surface.

Example 15 Identification of TYR-22 and TYR-131 as PossiblePhospho-Acceptors

Human MD-2 contains nine tyrosine residues (FIG. 14A). The inventorsmutated all nine tyrosine residues conservatively to phenylalanine andtested their ability to respond to LPS. HEK293 cells stably transfectedwith a NF-κB reporter gene and TLR4 were transiently transfected withplasmids encoding wild-type MD-2, MD-2-Y22F, MD-2-Y34F, MD-2-Y36F,MD-2-Y42F, MD-2-Y65F, MD-2-Y75F, MD-2-Y79F, MD-2-Y102F, or MD-2-Y131F.24 hrs later, cells were stimulated with LPS. As can be seen in FIG.14B, upon LPS stimulation the mutant proteins MD-2-Y22F and MD-2-Y131Fare significantly less potent in their ability to activate NF-κBcompared to wild-type MD-2. Analysis of a double mutant protein,MD-2-Y22F and Y131F, further confirmed that these residues are importantfor MD-2 to signal NF-κB activation in response to LPS (FIG. 14C). Itwas also determined that the mutant proteins lacking tyrosine residueslocated at positions 22 and 131 had a diminished ability to activateIL-8 (FIG. 14D). The inventors further characterized these mutant MD-2proteins by analyzing their phosphorylation status after LPSstimulation. Supporting the prediction that the sites mutagenized werephosphorylation sites, the mutant proteins were indeed lessphosphorylated compared to normal MD-2 (FIG. 14E). Additionally, theamount of phosphorylated p38 after LPS stimulation was measured. Cellstransfected with the mutant MD-2 proteins had less phosphor-p38 comparedto the normal MD-2 transfected cells, indicating dysfunctional signalingin these cells (FIG. 14E). In addition, it was confirmed that thesemutant proteins were secreted (data not shown), and that they displayeda similar glycosylation pattern as wild-type MD-2 upon SDS-PAGEanalysis, and that they were expressed at similar levels (FIG. 14E).

Given that it has been shown that Tyr 22 and Tyr 131 are possiblephospho-accepting residues, the inventors next determined the locationof these residues with respect to the published crystal structure ofMD-2. As shown in FIGS. 15 A and C, the hydroxyl-groups of both residuesappear to be surface exposed, thereby allowing phosphorylation of thesetyrosine residues to occur. Although Tyr 131 is located at thehydrophobic pocket of MD-2, neither Tyr 22 nor 131 are involved in themain dimerization interface of the TLR4-MD-2-LPS complex (FIG. 15, B andD).

In conclusion, two of the substitutions, MD-2-Y22F and MD-2-Y131F,resulted in a 50% decrease in NF-κB activity upon LPS stimulation,indicating that these residues were critical for maximal NF-κBactivation and can be phospho-accepting residues. In addition, byanalyzing the published crystal structure of MD-2, the inventorsdetermined that the hydroxyl groups of both MD2 tyrosine residues,located at positions 22 and 131, are surface exposed thereby permittingphosphorylation of the aforementioned residues to occur.

TABLE 4 Y to F sequences of human MD-2. Y22FMLPFLFFSTLFSSIFTEAQKQFWVCNSSDASISYTYCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 17)Y34F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISFTYCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 18)Y36F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTFCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 19)Y42F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYCDKMQFPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 20)Y65F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYCDKMQYPISINVNPCIELKRSKGLLHIFFIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 21)Y75F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLFFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 22)Y79F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLFITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 23)Y102F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDFSFCRALKGETVNTTISFSFKGIKFSKGKYKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 24)Y131F MLPFLFFSTLFSSIFTEAQKQYWVCNSSDASISYTYCDKMQYPISINVNPCIELKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTISFSFKGIKFSKGKFKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 25)Y22F MLPFLFFSTLFSSIFTEAQKQFWVCNSSDASISYTYCDKMQYPISINVNPCIE +LKRSKGLLHIFYIPRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFC Y131FRALKGETVNTTISFSFKGIKFSKGKFKCVVEAISGSPEEMLFCLEFVILHQPN SN (SEQ ID NO: 26)

Example 16 MD-2 was Shown to Interact with Lyn

Prior studies have shown that the Src kinase, Lyn, is recruited to TLR4(Medvedev et al. (2007) J BIOL CHEM 282, 16042-16053) as well as to CD14(Stefanova et al. (1993) J BIOL CHEM 268, 20725-20728). Furthermore ithas been suggested that Lyn may be involved in TLR4 tyrosinephosphorylation. Similar to previous results, the inventors found thatTLR4 immunoprecipitated with Lyn upon LPS stimulation (FIG. 16A). Giventhat MD-2 tyrosine phosphorylation was abolished following pretreatmentwith herbimycin A (FIG. 13B), a potent Src kinase inhibitor, which hasbeen shown to inhibit Lyn activity (June et al. (2006) PROC NATL ACADSCI USA 87, 7722-7726), Lyn involvement in the phosphorylation of MD-2was investigated. HEK293 cells were transiently transfected withMyc-TLR4, HA-MD-2, Flag-Lyn and CD14 constructs. HA-tagged proteins wereimmunoprecipitated from cell lysates with an anti-HA antibody andimmunoblotted with an anti-Flag antibody. The inventors observed thatLyn immunoprecipitated with MD-2 (FIG. 16B). However, given that Lynalso immunoprecipitates with TLR4, the inventors examined whether theinteraction between MD2 and Lyn was due to a direct interaction. HEK293cells were transiently transfected with plasmids expressing HA-MD-2 andFlag-Lyn. HA-tagged proteins were then immunoprecipitated andsubsequently immunoblotted with an anti-Flag antibody. The inventorsdiscovered that even without the presence of TLR4, MD-2immunoprecipitated with Lyn, thus confirming that Lyn and MD-2 coulddirectly interact (FIG. 16C). This result thus suggested that Lyn is themost likely kinase that phosphorylates MD2.

Example 17 Lyn Peptide Inhibitor Diminished MD-2 TyrosinePhosphorylation

Given that MD-2 tyrosine phosphorylation was abolished followingpretreatment with herbimycin A, and that MD-2 and Lyn were found to bein a complex together, the inventors confirmed that Lyn was required forMD-2 tyrosine phosphorylation. A Lyn specific peptide inhibitor (Adachiet al. (1999) J IMMUNOL 163, 939-946) was used to examine the effect ofinhibiting Lyn activation on MD-2 phosphorylation. HEK 293 cells,overexpressing TLR4, Myc-MD-2 and CD14 were pretreated with the Lynpeptide inhibitor for two hours prior to LPS stimulation. As can be seenin FIG. 17A, the Lyn peptide inhibitor significantly abolishedLPS-induced MD-2 tyrosine phosphorylation in a dose dependent manner(FIG. 17A, compare lanes 4-6 to lane 3). Furthermore, followingpretreatment with the Lyn peptide inhibitor, LPS-induced activation ofIL-8 and NF-κB was abolished in HEK293 cells overexpressing TLR4, CD14and MD2 (FIG. 17B), thus confirming the important role for Lyn kinase inmediating the signaling of the LPS-MD-2-TLR4-complex.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. It will be understood by those within the art that,in general, terms used herein are generally intended as “open” terms(e.g., the term “including” should be interpreted as “including but notlimited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.).

What is claimed is:
 1. A method of inhibiting lipopolysaccharide (“LPS”)activated toll-like receptor 4 signaling (“TLR4”), inhibiting LPSsignaling, or inhibiting LPS induced inflammation in a subject in needthereof, comprising: providing a purified polypeptide comprising anamino acid sequence that is at least 80% identical to SEQ ID NO: 1 orSEQ ID NO:2; and administering the polypeptide to the subject to inhibitLPS activated TLR4 signaling, inhibit LPS signaling, or inhibit LPSinduced inflammation.
 2. The method of claim 1, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:1.
 3. The method of claim2, wherein the polypeptide is glycosylated.
 4. The method of claim 1,wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:1and 1-20 conservative amino acid substitutions.
 5. The method of claim1, wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:1 and 1-20 amino acid insertions, deletions and/or substitutions. 6.The method of claim 1, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:2.
 7. The method of claim 6, wherein thepolypeptide is glycosylated.
 8. The method of claim 1, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:2 and 1-20conservative amino acid substitutions.
 9. The method of claim 1, whereinthe polypeptide comprises the amino acid sequence of SEQ ID NO:2 and1-20 amino acid insertions, deletions and/or substitutions.
 10. Themethod of claim 1, wherein LPS activated TLR4 signaling is inhibited.11. The method of claim 1, wherein LPS signaling is inhibited.
 12. Themethod of claim 1, wherein LPS induced inflammation is inhibited. 13.The method of claim 1, wherein the LPS induced inflammation is lunginflammation and the lung inflammation is inhibited.